The use triplex forming oligonucleotides (TFO) to sequence specifically bind to DNA is an attractive strategy in the treatment of diseases associated with aberrant or foreign gene expression. The ability to sequence specifically deliver agents into the major groove using TFOs has not been possible because the formation of triplex structures is restricted to extended homopurine sequences. This is a consequence of the: (i) asymmetric location of the TFO's sugar-phosphate backbone in the major groove; and (ii) change in polarity as the TFO reads information on the complementary strands. The result is that any interruption in a homopurine stretch causes a significant decrease in the TFO's binding affinity. It is hypothesized that these limitations can be overcome using TFO's with unnatural C-glycosides (oligoTRIPs) that will bind via Hoogsteen H-bonding to DNA with their glycosidic bond in the center of the major groove and perpendicular to the Watson-Crick duplex H-bonds. For each duplex pairing scheme (antiGC, 2-amino-quinolin-4-yl; antiCG, 2-amino-quinolin-5-yl; antiAT, 2-amino-quinazolin-4-yl; antiTA, 2-amino-quinazolin-5-yl) there is a unique TRIP base. The location of the sugar phosphate backbone and the novel heterocyclic bases are designed to allow the oligoTRIPs to bind to mixed purine/pyrimidine sequences and "read DNA in a single direction. The Specific Aims of the proposal are to: (1) Synthesize and characterize C-glycosides, referred to as TRIPs, that can be readily assembled into oligomers (oligoTRIPs) that are designed to sequence specifically bind in the major groove of duplex Watson-Crick DNA with via a triple helix motif and without a requirement for homopurine sequences or non-physiological pH. (2) Evaluate the in vitro binding affinities and specificities of the oligoTRIPs using gel shift assays, TM measurements, and chemical and enzymatic footprinting. (3) Characterize the molecular forces that influence the stability and structure of DNA triplexes with oligoTRIPs and quantify the thermodynamics governing triple helical formation, including the role of sequence, cations and hydration. (4) Append an Fe-EDTA DNA cleaving functionality on the 5' or 3'-terminus of the oligoTRIPs and determine if these molecules generate sequence specific damage in large DNA targets; and (5) Perform high resolution NMR studies on triplexes formed between oligoTRIPS and duplex DNA.